The present invention relates to polysialic acid derivatives which are useful for conjugation to drugs, proteins and peptides, or to drug delivery systems such as liposomes, having sulfhydryl groups, as well as to conjugated products process for synthesising the derivatives and the conjugates and novel synthetic intermediates.
The extended presence of drugs either within the vascular system or in extravascular use is often a pre-requisite for their optimal use. Many antibiotics and cytostatics for instance, as well as a variety of therapeutic peptides and proteins, and liposomes are removed from the circulation prematurely and before effective concentrations in target tissues can be achieved. The half lives of a number of short-lived proteins (for instance enzymes, cytokines, etc.) have been augmented by conjugating these to low poly(ethylene glycol). It appears that PEG molecules prolong the circulation time of proteins and particles by forming a cloud around their surface, thus sterically hindering interaction with factors responsible for their clearance. However PEG is non-biodegradable and accumulation of PEGylated proteins intracellularly may be undesirable especially on chronic use (Bendele, A., Seely, J., Richey, C., Sennello, G., Shopp, G., Renal tubular vacuolation in animals treated with polyethylene-glycol conjugated proteins, Toxicological sciences, 42 (1998) 152-157; Conyers, C. D., Lejeune, L., Shum, K., Gilbert, C., Shorr, R. G. L, Physiological effect of polyethylene glycol conjugation on stroma-free bovine hemoglobin in the conscious dog after partial exchange transfusion, Artificial organ, 21 (1997) 369-378).
We have described the conjugation of a polysaccharide comprising 2→8 and or 2→9 (e.g., alternating 2→8 and 2→9) linked sialic acid units conjugated to proteins to increase their half life, reduce their immunogenicity/antigenicity or increase the stability of a variety of proteins. In WO92/22331, polysialic acids are reacted with a model drug and shown to extend the half life in the circulation of mice. In Cell. Mot. Life Sci. 57 (2000) 1964-1969 and in Biotechnol. Genet. Eng. Rev. 16 (1999) 203-215, Gregoriadis, et al., describe the conjugation of polysialic acids to asparaginase and catalase, and show that the clearance rates from circulation reduced whilst enzyme activity was retained. We have also polysialylated insulin (Biochim. Biophys. Acta 1622 (2003) 42-49 and shown it to be active. We have also polysialylated interferon (AAPS Annual meeting 2002, Toronto, Canada, M1056). We have also polysialylated antibody fragment Fab (Epenetos, A., et al., Proceedings of ASCO (Clinical Pharmacy) 21 (2002) 2186).
In all of these publications, polysialic acid is rendered reactive, by generating an aldehyde group at the non-reducing end by oxidation of the 7,8-vicinaldiol moiety with sodium periodate. The aldehyde group was then reacted with primary amine groups on proteins, generally assumed to be epsilon-amino groups of lysine moieties of the protein or N-terminal amine groups. The reaction forms a Schiff base which is reduced by cyanoborohydride to a secondary amine.
In WO-A-01/87922 we also suggest that derivatisation with other molecules could be carried out in the presence of denaturant to achieve increased levels of derivatisation. Examples of other derivatising agents are polyethylene glycol compounds. Activated PEG compounds such as tresyl-PEG and succinimidyl succinate ester of PEG were mentioned. The examples used succinimidyl succinate activated PEG, which is believed to react with amine groups.
PEG derivatives having functional groups for coupling to thiol groups are commercially available. The functional groups may be maleimide, vinyl sulfone, iodoacetamide or orthopyridyl disulphide groups. Since these reagents react specifically with cysteines, and since proteins have fewer cysteines on their surfaces than lysine groups, the derivatisation is more controllable. Furthermore, in the absence of a free cysteine in a native protein, one or more free cysteines may be added by genetic engineering. The advantage of this approach is that it makes possible site-specific derivatisation at areas on the protein which will minimise a loss in biological activity.
PEGylated proteins have been found to generate anti PEG antibodies that could also influence the residence time of the conjugate in the blood circulation (Cheng T, Wu, M., Wu, P., Chem, J, Roffer, S R., Accelerated clearance of polyethylene glycol modified proteins by anti-polyethylene glycol IgM, Bioconjugate chemistry, 10 (1999) 520-528. Despite, the established history of PEG as a parenterally administered polymer conjugated to therapeutics, a better understanding of its immunotoxicology, pharmacology and metabolism will be required (Hunter, A. C, Moghimi, S. M., Therapeutic synthetic polymers: a game of Russian Roulette. Drug Discovery Today, 7 (2002) 998-1001; Brocchini, S., Polymers in medicine: a game of chess. Drug Discovery Today, 8, (2003) 111-112).
It would be useful for modification by polysialic acid to be targeted towards thiol (sulfhydryl) groups. It would also be desirable for the efficiency of derivatisation by sialic acid to be increased, the processes described in our prior art mentioned above requiring high excesses of active polysialic acid. It would also be desirable to avoid the use of cyano borohydride.